Solid-phase chemical analysis using array hybridization facilitated by agitation during certrifuging

ABSTRACT

Array hybridization can be facilitated by agitating a reaction cell subject to centrifugal force greater than 1G. A two-dimensional hybridization array is preferably oriented generally orthogonal to the centrifugal force. Agitation involves titling the array back and forth about an axis, preferably parallel to a centrifuge axis. The centrifugal force serves, in a sense, as supergravity helping to overcome non-specific binding forces (viscous forces and other forces at the liquid-solid boundary) that limit the rate of liquid flow. Thus, the agitation rate and the related replenishment rate can be increased. The agitation causes the sample liquid to wash back and forth across the array, which remains protected by a thin liquid film. The resulting “tidal” motion, results in thorough mixing of the sample liquid. In addition, since only a thin film is required over much of the array, typically costly sample volume can be reduced. Thus, faster hybridization with lower sample volumes can be achieved.

BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to solid-phase chemistry and, more particularly, to chemical and biochemical reactions between a molecules bound to a substrate surface and molecules in a liquid, as in array hybridization. A major objective of the present invention is to provide for more rapid array hybridization.

[0002] Solid-phase chemistry involves chemical or biochemical reaction between components in a fluid and molecular moieties present on a substrate surface. Solid-phase chemical reactions can involve: the synthesis of a surface-bound oligonucleotide or peptide, the generation of combinatorial “libraries” of surface-bound molecular moieties, and hybridization assays in which a component present in a fluid sample hybridizes to a complementary molecular moiety bound to a substrate surface.

[0003] Hybridization reactions between surface-bound molecular probes and target molecules in a sample liquid may be used to detect the presence of particular biomaterials including biopolymers and the like. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules coverable of reacting with target molecules in solution. Such reactions form the basis for many of the methods and devices used in the new field of genomics to probe nucleic acid sequences for novel genes, gene fragments, gene variants and mutations.

[0004] The ability to clone and synthesize nucleotide sequences has led to the development of a number of techniques for disease diagnosis and genetic analysis. Genetic analysis, including correlation of genotypes and phenotypes, contributes to the information necessary for elucidating metabolic pathways, for understanding biological functions, and for revealing changes in genes that confer disease. New methods of diagnosis of diseases, such as AIDS, cancer, sickle cell anemia, cystic fibrosis, diabetes, muscular dystrophy, and the like, rely on the detection of mutations present in certain nucleotide sequences. Many of these techniques generally involve hybridization between a target nucleotide sequence and a complementary probe, offering a convenient and reliable means for the isolation, identification, and analysis of nucleotides.

[0005] In biological chip or “biochip” arrays, a plurality of probes, at least two of which are different, are arranged in a spatially defined and physically addressable manner on a substrate surface. Such “biochip” arrays have become an increasingly important tool in the biotechnology industry and related fields, as they find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. Substrate-bound biopolymer arrays, particularly oligonucleotide, DNA and RNA arrays, may be used in screening studies for determination of binding affinity and in diagnostic applications, e.g., to detect the presence of a nucleic acid containing a specific, known oligonucleotide sequence.

[0006] Regardless of the context, all chemical or biochemical reactions between components in a fluid and molecular moieties present on a substrate surface require that there be adequate contact between the fluid's components and the surface-bound molecular moieties. To this end, a number of approaches have been proposed to facilitate mixing of fluid components during solid phase chemical or biochemical reactions so that a substantially homogeneous fluid contacts the reactive surface. Most recently, a great deal of attention has focused on improving hybridization assays using various mixing techniques.

[0007] Inadequate mixing is a particular problem in chemical and biological assays where very small samples of chemical, biochemical, or biological fluids are typically involved. Inhomogeneous solutions resulting from inadequate mixing can lead to poor hybridization kinetics, low efficiency, low sensitivity, and low yield. With inadequate mixing, diffusion becomes the only means of transporting the reactants in the mobile phase to the interface or surface containing the immobilized reactants. In such a case, the mobile phase can become depleted of reactants near the substrate as mobile molecules become bound to the immobile phase. Also, if the cover is not exactly parallel to the plane of the substrate, the height of the fluid film above the probe array will vary across the array. Since the concentration of target molecules will initially be constant throughout the solution, there will be more target molecules in regions where the film is thicker than in regions where it is thinner, leading to artifactual gradients in the hybridization signal.

[0008] As array density is ever increasing, and the need for faster and more accurate hybridization assays is ongoing, there is currently a great deal of emphasis on improving “mixing” of sample liquid during hybridization and, correspondingly, in maximizing contact between the components of the sample liquid and the entirety of the array surface.

[0009] The Affymetrix GeneChip® Fluidics Station hybridization and wash instrument includes a means for pumping a sample liquid back and forth across an array on a substrate surface while the substrate is mounted in a holder. While this method provides for mixing of components within the sample liquid, there are disadvantages that can adversely affect the accuracy of the hybridization reaction. That is, the method is prone to contamination because of the number and variety of materials that come into contact with the sample liquid, i.e., adhesives, various plastic components, and the like. In addition, large sample volumes (greater than 200 microliters (μl) are required, and temperature control is poor.

[0010] In U.S. Pat. No. 4,849,340 to Oberhardt, an alternative means is disclosed for mixing components in a fluid during an assay performed in an enclosed chamber. Oberhardt discloses an apparatus comprising a base, an overlay and a cover which when combined define a sample well, a channel, and a reaction space. Fluids introduced into the sample well flow by coverillary action to the reaction space. Mixing of fluids within the reaction space is effected using mechanical or electromechanical means to create forced convection currents. Again, large sample volumes are required (100 to 200 μl) because of the need to maintain a gap between the base and the cover during mixing. Additionally, the method relies on coverillary action to promote fluid flow, and mixing may thus be slow and incomplete, particularly when viscous reagents are used.

[0011] U.S. Pat. No. 5,192,503 to McGrath et al. discloses an apparatus for conducting an in situ assay of a tissue section mounted on a slide. A seal member, mounted on a plate, forms a closed periphery and encloses and defines an interior region on the slide that forms a reaction chamber. A plate covers the slide and seal member. The joined plate and slide together form a probe clip. The reaction chamber may comprise a single chamber or two chambers. In the one-chamber embodiment a time-release material, such as gelatin, is applied over the probe, allowing time for reaction of the tissue sample with reagents before the probe is released and thus able to react with the tissue sample.

[0012] In the two-chamber embodiment, the probe reaction chamber defined by the closed periphery of a first seal member is divided into two regions by a raised portion of the plate, a mixing chamber and a reaction chamber. At least one end of this raised portion does not contact the first seal member, thereby leaving a channel available for fluid flow. Probe compounds placed in the mixing chamber do not mix with the fluid reagents in the reaction chamber until fluid is induced to flow between the two chambers via a channel in a gap left between the raised portion and the seal member. Fluid flow may be induced by rotating the probe clip to a substantially vertical orientation, allowing fluid reagents from the reaction chamber to flow into the mixing chamber and mix with the probe compounds. Re-orienting the probe clip to the horizontal causes the mixed probe and fluid reagent to flow to the reaction chamber for reaction with a tissue section therein. Thus, the position and flow of fluid reagents and probes in the reaction chamber and the mixing chamber is controlled by gravity. Optionally, both gravity-controlled flow and use of a time-release agent such as gelatin may be used at the same time to regulate the mixing of reagent fluids and probes. Like the Oberhardt device, the McGrath et al. apparatus is disadvantageous when viscous solutions are used or rapid mixing is required, insofar as mixing depends upon gravity to induce flow.

[0013] A method for more thorough mixing of components in a sample liquid during a solid phase chemical or biochemical reaction is disclosed in commonly assigned, co-pending U.S. patent application Ser. No. 09/343,372 to Schembri et al., filed Jun. 30, 1999 (“Apparatus and Method for Conducting Chemical or Biochemical Reactions on a Solid Surface Within an Enclosed Chamber”). That method involves mixing a very thin film of fluid in a chamber, wherein an air bubble is incorporated therein and, when used in hybridization, a surfactant is preferably present as well. However, non-specific binding between the sample liquid and reaction cell surfaces, as well as liquid viscosity limit the rate the air bubble can be moved in the reaction cell. This in turn limits the rate at which mixing can occur, and thus the hybridization rate.

[0014] In FIG. 3, reaction cell 30 is being rotated counterclockwise as indicated by arrows 50. Centrifugal force arrows 45 are shown broken into a component 51 along array 40 and a component 53 orthogonal to array 40. The component 51 along array 40 urges sample liquid 39 toward (left) end 47. This movement is indicated by arrows 55, which show a tapered liquid profile flowing toward (left) end 47. A curved arrow 47 shows a return motion for liquid sample 39. This return motion provides for highly desirably vertical mixing.

[0015] The vertical mixing assures that every target molecule spends some time close enough to array 40 for binding to occur. The centrifugal force 45 helps overcome the inertia of the liquid and its non-specific binding forces with the substrate so that a high agitation rate can be maintained. The advantages of the invention can be understood with the following, admittedly approximate, understanding of the hybridization process.

[0016] When the agitation rate is doubled, each target molecule is likely to be found half as far from a respective probe for half the time. When it is half as far, it is four times as likely to hybridize. However, the interval over which it can hybridize is half as long. Thus, in principle, doubling the agitation rate doubles the hybridization rate. This linear relationship applies until non-specific binding fluid forces prevent sample liquid from completing its motion across the array. The stronger the centrifugal force, the higher the agitation rate can be raised before this limiting consideration applies. Thus, the centrifuge rate can be increased until the forces involved adversely affect specific binding or threaten the integrity of the hybridized or non-hybridized species.

[0017] In FIG. 1, the agitation axes are parallel to the centrifuge axis and the hybridization arrays are generally orthogonal to the centrifugal force. In other embodiments, the hybridization arrays are also generally orthogonal to the centrifugal force, but the agitation axes are not parallel to the centrifuge axis. For example, the agitation axes can be circumferentially (in other words, “tangentially”) oriented relative to the centrifuge axis.

[0018] Particularly with a circumferentially oriented agitation axis, but also other cases in which the array is orthogonal to the centrifugal force, the substrate can be curved cylindrically, for example, along a radius slightly less than (e.g., 90% of) the distance between the agitation axis and the centrifuge axis. In this case, the centrifugal force is more orthogonal to the array away from the array center and even at the extremes of the agitation motion. This provides a more uniform sample liquid distribution across the array, which in turn allows less sample liquid to be used without risking drying of the array. In addition, the agitation is gentler on the sample.

[0019] Reaction cells 30 of FIG. 1 are oriented so that arrays 40 generally orthogonal to the centrifugal force. Oblique orientations are also provided for. For example, reactions cells can be oriented so that they are more orthogonal to the centrifugal force than along it. However, reaction cells 530 of FIG. 4 represent another case in which reaction cells are oriented both along and orthogonal to the centrifugal force.

[0020]FIG. 4 shows system AP1 with reaction cells 530 oriented parallel to turntable 17. Reaction cells 530 are similar to reaction cells 30 and likewise include a probe array, in this case, probe array 540. Centrifugal force 545 urges sample liquid 549 radially outward, so that gas with cell 530 is radially inward of liquid 539. In this case, the agitation axis is perpendicular and through the center of array 540.

[0021] In greater detail with reference to FIG. 3, reaction cell 30 includes substrate 31 that preferably has a substantially planar surface, with at least a portion of the surface representing a reaction area (hybridization array 40) on which the chemical or biochemical reactions are conducted, and cover 33, optimally of plastic, having a peripheral lip which sealingly contacts the substrate surface about the reaction area, and wherein the cover and the reaction area of the substrate surface form an enclosure having an interior space that serves as the reaction chamber. The chamber is adapted to retain a quantity of fluid so that the fluid is in contact with the reaction area of the substrate surface and the inner surface of the cover.

[0022] The reaction cell also includes a fastening means (not shown) effective to press the cover and the substrate together, i.e., to immobilize the cover on the substrate, thereby forming a watertight, temporary seal therebetween. The fastening means ensures stable, effective and secure positioning of the cover over the substrate. Optional gasket means adjacent the surface of the cover may be included to aid in equalizing the pressure provided by the fastening means. The optional gasket may be, for example, placed between the cover and the rigid frame to provide compliance in the system and to even the pressure applied to the cover and the substrate. The apparatus further comprises fluid transfer means which enables introduction of fluid from the exterior of the apparatus to the reaction chamber, and removal therefrom. In a preferred embodiment, the fluid introduction means comprises one or more ports in the cover.

[0023] It is preferred that the cover be made of plastic and the substrate of glass, plastic, fused silica or silicon, the seal between plastic and either glass, plastic, fused silica or silicon being advantageous for producing the apparatus of the invention. The cover material should be thermally stable, chemically inert, and preferably non-stick. Furthermore, when the apparatus is used in hybridization, the cover should be comprised of a material that is chemically and physically stable under conditions employed in hybridization. In a preferred embodiment, the plastic cover is polypropylene, polyethylene or acrylonitrile-butadiene-styrene (“ABS”). In the most preferred embodiment, the plastic cover is comprised of polypropylene. The cover may be constructed by machining or molding technologies.

[0024] As noted above, the cover preferably has a lip along the perimeter of the cover bordering a recessed portion that comprises the major portion of the area of the inner face of the cover. Applying pressure to the outer face of the cover directly above the perimeter lip is required to form the tight seal between the cover and the substrate. Any means that presses the lip of the cover securely to the substrate is suitable. Such pressure may be applied evenly by, for example, clamps, a press, or by coverturing the substrate and cover within a two-part rigid frame and compressing the two together to supply an even pressure to the cover and substrate. If desired, the peripheral lip of the cover may be modified to provide for an improved seal; for example, one or more continuous ridges can be incorporated into the lip so that the pressure supplied to the cover is higher at those locations and preferentially causes them to compress. In any of these embodiments, the reaction cell may be re-used, as the peripheral seal is temporary and the fastening means may be removed when desired. Thus, the reaction cell may be readily disassembled after use, cleaned, and re-assembled (with alternate components, such as a different substrate, if desired) so that some or all of the components of the reaction cell may be re-used.

[0025] This reaction cell interior height may range from about 0.002″ to 0.02″ (50 μm to 500 μm). The dimension of the cover, the peripheral lip, and the reaction area are such that the reaction area is generally in the range of about 4 mm² to 500 mm², preferably about 20 mm² to 350 mm², and the reaction chamber has a volume in the range of about 0.2 μl to about 312 μl, preferably about 1 μl to 200 μl.

[0026] Hybridization array 40 has a plurality of molecular probes bound thereto. Preferably, the molecular probes are arranged in a spatially defined and physically addressable manner, i.e., are present in one or more “arrays.” In a most preferred embodiment, the probes are oligonucleotide probes (including cDNA molecules or PCR products), although other biomolecules, e.g., oligopeptides and the like, may serves as probes as well.

[0027] The term “hybridization” as used herein means binding between complementary or partially complementary molecules, as between the sense and anti-sense strands of double-stranded DNA. Such binding is commonly non-covalent binding, and is specific enough that such binding may be used to differentiate between highly complementary molecules and others less complementary. Examples of highly complementary molecules include complementary oligonucleotides, DNA, RNA, and the like, which comprise a region of nucleotides arranged in the nucleotide sequence that is exactly complementary to a probe; examples of less complementary oligonucleotides include ones with nucleotide sequences comprising one or more nucleotides not in the sequence exactly complementary to a probe oligonucleotide.

[0028] For use in hybridization, the interior of the reaction cell, in other words, the “hybridization chamber,” is filled with a sample liquid comprising a target molecule which may hybridize to a surface-bound molecular probe, and with a surfactant of a type and present at a concentration effective to substantially reduce nonspecific binding and promote mixing of components within the sample liquid. The surfactant is selected from the group consisting of anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, and combinations thereof, with anionic surfactants and polymeric nonionic surfactants particularly preferred. Suitable anionic surfactants include, but are not limited to, the sodium, potassium, ammonium and lithium salts of lauryl sulfate, with lithium lauryl sulfate most preferred. A preferred polymeric nonionic surfactant is polyethylene oxide, with particularly preferred polyethylene oxides comprising an alkylphenol ethylene oxide condensate. Such surfactants may be obtained commercially under the trade name “Triton” from the Sigma Chemical Company (St. Louis, Mo.), and including, for example, Triton X-100 (octylphenol ethylene oxide condensate) and Triton X-102 (also an octylphenol ethylene oxide condensate). More specifically, Triton X surfactants have been described as having the formula:

[0029] in which N for Triton X-100 has an average of about 9.5 units per molecule while for Triton X-102 N is an average of about 12.5 units per molecule. Further information on both Triton X-100 and Triton X-102 can be found at the following Internet addresses: “www.sigma-aldrich.com/sigma/proddata/t6878.htm” and “www.sigma-aldrich.com/sigma/proddata/t6878x.htm”.

[0030] The surfactant generally represents between about 0.1 wt. % and 10 wt. % of the sample liquid, preferably between about 0.5 wt. % and 5 wt. % of the sample liquid, more preferably between about 0.75 wt. % and 5 wt. % of the sample liquid; however, it should be emphasized that the exact concentration will vary with the surfactant selected, and those skilled in the art may readily optimize the concentration with respect to the desired results, i.e., reduction of nonspecific binding and facilitation of mixing within the sample liquid. An exemplary sample liquid will contain between about 0.1 wt. % and about 1 wt. % of polyethylene oxide and between about 0.05 wt. % and about 1 wt. % lithium lauryl sulfate.

[0031] The invention is particularly useful in conjunction with substrate surfaces functionalized with silane mixtures, as described in co-pending, commonly assigned U.S. patent application Ser. No. 09/145,015, filed Sep. 1, 1998, and entitled “Functionalization of Substrate Surfaces with Silane Mixtures.” That method provides a functionalized surface on a substrate with low surface energy. The method for preparing such a surface comprises contacting a substrate having reactive hydrophilic moieties on its surface with a derivatizing composition comprising silane-containing groups R¹-Si(R^(L)R^(x)R^(y)) and R²-(L)_(n)—Si(R^(L)R^(x)R^(y)) under reaction conditions effective to couple the silanes to the substrate. This provides —Si-R¹ and —Si—(L)_(n)-R² groups on the substrate. The R^(L), which may be the same or different, are leaving groups, the R^(x) and R^(y) which may also be the same or different, are either leaving groups, like R^(L), or are lower alkyl, R¹ is a chemically inert moiety that upon binding to the substrate surface lowers the surface energy thereof, n is 0 or 1, L is a linking group, and R² comprises either a functional group enabling covalent binding of a molecular moiety or a group that may be modified to provide such a functional group.

[0032] The ratio of the silanes in the derivatizing composition determines the surface energy of the functionalized substrate and the density of molecular moieties that can ultimately be bound to the substrate surface. When used in conjunction with the present invention, the surface-bound molecular probes are bound to the R² moieties provided by the second silane-containing group. These and other variations upon and modifications to the disclosed embodiments are provided for by the present invention, the scope of which is defined by the following claims. 

What is claimed is:
 1. An apparatus for array hybridization comprising: a reaction cell for confining sample liquid and a gas, said reaction cell having an array of probes for hybridizing with respective complementary molecular components of said liquid sample; a centrifuge for rotating said reaction cell so as to impose a centrifugal force greater than 1 G on said sample liquid, said centrifuge having a rotor that rotates about a centrifuge axis; an agitator for rotating said reaction cell relative to said rotor so that said sample liquid moves relative to said array, said agitator being mechanically coupled to said centrifuge and said reaction cell.
 2. An apparatus as recited in claim 1 wherein said agitator rotates said reaction cell about an axis more orthogonal to than along said centrifugal force.
 3. An apparatus as recited in claim 2 wherein said agitator changes direction of rotation of said reaction cell relative to said rotor periodically so as to define an agitation cycle rate.
 4. An apparatus as recited in claim 3 wherein said rotor has a rotation rate greater than said agitation cycle rate.
 5. An apparatus as recited in claim 2 wherein said agitation means rotates said reaction cell about an axis that extends more parallel to said centrifuge axis than orthogonal to it.
 6. An apparatus as recited in claim 5 wherein said array extends more orthogonal to than parallel to said centrifugal force so that said centrifugal force forces said sample liquid against said array.
 7. An apparatus as recited in claim 6 wherein said agitation means rotates said reaction cell about said agitation axis so that said centrifugal force forces liquid in said reaction cell away from said array.
 8. An array hybridization method comprising the steps of: introducing sample liquid into a reaction cell so that some interior volume is partially occupied by sample liquid and partially occupied by gas; centrifuging said sample liquid by rotating said reaction cell having a probe array so that centrifugal forces urges said sample liquid against said array; and agitating said sample liquid in said reaction cell during said centrifuging so that said sample liquid moves relative to said array.
 9. An array hybridization method as recited in claim 8 wherein said agitation involves rotating said sample cell about an agitation axis that is more orthogonal to than along said centrifugal force.
 10. An array hybridization method as recited in claim 9 wherein said agitating involves periodically changing the direction of rotation about said agitation axis so as to define an agitation cycle rate.
 11. An array hybridization method as recited in claim 11 wherein said centrifuging involves rotating said reaction cell at a centrifuge rate greater than said agitation rate.
 12. An array hybridization method as recited in claim 10 wherein said agitation involves rotating said sample cell about an agitation axis that extends more parallel to said centrifuge axis.
 13. An array hybridization method as recited in claim 12 wherein said array extends more orthogonal to centrifugal than along it so that said centrifugal forces urges said sample liquid against said array.
 14. An array hybridization method as recited in claim 13 further comprising a step of removing sample liquid from said reaction cell, said removing step involving rotating said reaction cell by rotating it about said agitation axis so that said centrifugal force urges said fluid in said reaction cell away from said array.
 15. An array hybridization method as recited in claim 8 wherein said reaction cell is filled at most half way with sample liquid. 